Question

The Sanger method for N-terminus determination is a less common alternative to the Edman degradation. In the Sanger method, the peptide is treated with the Sanger reagent, 2,4-dinitrofluorobenzene, and then hydrolyzed by reaction with 6 M aqueous HCl. The N-terminal amino acid is recovered as its 2,4-dinitrophenyl derivative and identified.

(a)Propose a mechanism for the reaction of the N terminus of the peptide with 2,4-dinitrofluorobenzene.

(b) Explain why the Edman degradation is usually preferred over the Sanger method.

Short answer

(a) Reaction of N-terminus of the peptide with 2,4-dinitrofluorobenzene is an example of nucleophilic aromatic substitution by addition-elimination mechanism.

(b) Sanger’s method can only analyse one amino acid per sample of protein whereas Edman’s method can analyse more than 20 amino acids per sample of protein.

Step-by-step solution

Step-1. Mechanism for the reaction of N-terminus of peptide with 2,4-DNFB:

Reaction of N-terminus of the peptide with 2,4-dinitrofluorobenzene is a nucleophilic aromatic substitution which takes place by addition-elimination mechanism. The nitrogen of N-terminus acts as a nucleophile and attacks at the electrophilic center to which fluorine is attached. As a result, carbanion intermediate is formed which undergoes resonance and then fluorine atom leaves as a leaving group which also acts as a base in next step and neutralises the positive charge on nitrogen which leads to formation of final product. Presence of two nitro groups makes this reaction favourable at mild conditions.

Mechanism of N-terminus of peptide with 2,4-DNFB reagent

Step-2. Drawback of Sanger’s method:

The main drawback of Sanger’s method is that it can only analyse one amino acid per sample of protein whereas, Edman’s method can analyse more than 20 amino acids per protein sample at a time. In Edman’s method, no pre-treatment of sample is required and identification from unknown proteins is possible that are not present in databases which is not possible in Sanger’s method.